1. Field of the Invention
The present invention relates to methods and to analytical systems for performing fibrinogen assays.
2. Discussion of The Background
Blood clotting reactions, in general, employed as clinical assays, measure the time required for the formation of a fibrin clot. Blood clotting assays are principally used for screening, diagnosis, and for monitoring patients receiving anticoagulant therapy.
There are many types of coagulation assays. These include: prothrombin time (PT); partial thromboplastin time (PTT); activated partial thromboplastin time (APTT); fibrinogen assay (i.e., the measurement of the concentration of clottable fibrinogen in a sample); thrombin time, also known as thrombin clotting time (TCT); activated clotting time (ACT); etc. The most frequently performed of these assays is prothrombin time.
The determination of the concentration of clottable fibrinogen in plasma is important for the investigation of coagulation disturbances in patients. Both immunological methods and coagulation tests have been used for the determination of fibrinogen. The immunological methods display severe diagnostic disadvantages and have consequently not achieved practical importance.
In coagulation tests, the fibrinogen content is determined by the time required for coagulum formation. The most important of these methods is the method of Clauss (see Acta Haemat. (1957) 17: 237-246).
In the Clauss method, a diluted plasma, i.e., a weak fibrinogen solution, is mixed with a concentrated thrombin solution, the amount of thrombin being about 550 U ml.sup.-1 of plasma. With the help of a calibration curve, the fibrinogen content of the sample is correlated to the time taken for the visible appearance of a coagulum. Coagulation tests in which one records photometrically the formation of turbidity during the course of coagulation are also known. See, e.g., Ratge et al, Clin. Chem. (1987) 33 (3): 420.
Finally, quantitative methods are also known in which the coagulum formed is isolated and its protein content determined. In this approach, the sample is reacted with thrombin and the coagulum formed isolated, washed and then dried. The protein content of the coagulum or its weight is then determined.
Becker et al (U.S. Pat. No. 4,692,406) disclose a method for the simultaneous determination of fibrinogen and of fibrinogen fission products in plasma. This method uses a snake venom enzyme with thrombin-like activity. In this method, the period of time between the addition of the enzyme and commencement of turbidity formation, which is a measure of the amount of fibrinogen fission products, is measured. The speed of turbidity formation is subsequently measured to determine the amount of fibrinogen present in the sample.
The prothrombin time test and the activated partial thromboplastin time test are each commonly used clinical tests to determine a patient's ability to form clots. These tests, and the other tests noted above are extensively used by hospitals, clinics, and laboratories for preoperative evaluations and for anticoagulant therapy administered to cardiac patients, among other patients. These tests are each based upon time measurements, and for the most part measure what is called an end point or clotting time, which occurs when fibrinogen is being polymerized to fibrin.
Many of these types of assays monitor change in sample optical density to measure the reaction. See, for example, Natelson et al (Am. J. Clin. Path. (1974) 61(6): 828-833), Lipscomb (U.S. Pat. No. 4,720,787), Saito et al (U.S. Pat. No. 4,217,107), Baughman et al (U.S. Pat. No. 4,289,498), Gross et al (U.S. Pat. No. 3,458,287), Eichelberger et al (U.S. Pat. No. 4,047,890), Becker et al (U.S. Pat. No. 4,692,406), Callahan et al, "Semiquantitative Fibrinogen Determination From the PT Clotting Reaction", Tech. Bulletin Tech. THR8804, copyright 1988 by Organon Teknika, Durham, N.C., USA, and Carroll et al "The Clot Signature and New Aspects in Coagulation Testing" July 1989, Ortho Diagnostic Systems Inc, Raritan, N.J., USA.
In addition to being assayed by the coagulation rate method as in the Clauss method noted above, fibrinogen can be assayed by the coagulation rate as in the Clauss method modified by Vermylen et al (Clin. Chem. Acta (1963) 8:418-424), or by sulfite precipitation, Rampling et al. (Clin. Chem. Acta (1976) 67:43), or by the total coagulable fibrinogen method of Ratnoff et al (J. Lab. Clin. Med. (1951) 37:316-320), or by an assay system based on the turbidity rate measurement of the conversion of fibrinogen to fibrin polymer sold by Du Pont (Du Pont Aca.TM., Du Pont Clinical Systems, Wilmington, Del. USA). The Vermylen et al method uses a glass hook or platinum loop which is continuously moved in and out of the clotting mixture until the appearance of a fibrin web as the end-point.
Today, there are 750,000 cases of acute myocardial infarction in the U.S. annually and more than one million combined cases of other arterial embolic events, pulmonary embolism (PE) and deep vein thrombosis (DVT). Approximately 25% to 35% of these cases are potential candidates for thrombolytic therapy. This therapy consisting of intravenous administration of a fibrinolytic drug to promote the dissolving of the occluding blood clot will be applied in many small community hospitals.
There currently exists a 0.5% rate of intracranial bleeding and from 1% to 20% rate of significant extracranial bleeding. At present, no convenient, reliable and rapid diagnostic instrument is available to follow the patient.
A challenge which is now emerging is how to better control thrombolytic therapy to maximize effectiveness and minimize risk of bleeding problems. Although no single diagnostic assay thus far appears to be the answer to this challenge, integration of a combination of diagnostic assays and other indicators could improve therapy.
Fibrinogen measurement, while difficult to achieve at the bedside accurately and conveniently, is an important parameter in thrombolytic therapy, particularly with regard to assessment of bleeding risk and therapeutic management of bleeding once it occurs. The measurement of initial fibrinogen drop, even that associated with fibrin selective agents, such as recombinant tissue plasminogen activator (rt-PA), would also be useful confirmation that the lytic process has begun. This is equally important for other fibrin selective agents such as streptokinase, urokinase, and anistreplase, since these drugs work by means of a systemic lytic effect.
With existing prior art methods for fibrinogen determination, centrifugation of the blood is necessary before performing the assay, because the blood cells interfere with the measurement. Separation of the blood cells takes time and increases the overall time required for the assay. If a fibrinogen assay can be performed as soon as the blood is collected, in vitro artifacts which arise from plasmin activation (due to the action of thrombolytic drugs) should minimally, if at all, affect test results.
These artifacts arise from the action of plasmin on a variety of proteins associated with blood coagulation including fibrinogen itself. This occurs in vitro after the blood sample has been collected. A delay of even several minutes (currently at least fifteen minutes with existing methods) produces inaccurate results. One solution to this problem has been to use inhibitors of plasmin or plasminogen activator as an additive to the blood collection tube to preserve the sample prior to testing. The use of inhibitors, however, adds additional expense and also restricts the field of functional assays which may be performed subsequently on the sample.
As noted supra in the past, with streptokinase, thrombin time testing had been employed to establish the presence of a lytic effect in DVT and PE where antibodies to streptokinase could neutralize a portion of the effect. A convenient, rapid, and accurate fibrinogen assay capable of being performed by the addition of one drop of whole blood to a dry chemistry test card would be a significant improvement in current diagnostic potential and could go a long way toward optimizing thrombolytic therapy for a particular patient. In addition, the diagnostic capacity of such a system could aid in clinical trials of the many newly emerging thrombolytic drugs currently in development. There is thus a clear need for such an assay.